A genome-wide copy number association study of osteoporotic fractures points to the 6p25.1 locus.

BACKGROUND: Osteoporosis is a systemic skeletal disease characterised by reduced bone mineral density and increased susceptibility to fracture; these traits are highly heritable. Both common and rare copy number variants (CNVs) potentially affect the function of genes and may influence disease risk. AIM: To identify CNVs associated with osteoporotic bone fracture risk. METHOD: We performed a genome-wide CNV association study in 5178 individuals from a prospective cohort in the Netherlands, including 809 osteoporotic fracture cases, and performed in silico lookups and de novo genotyping to replicate in several independent studies. RESULTS: A rare (population prevalence 0.14%, 95% CI 0.03% to 0.24%) 210 kb deletion located on chromosome 6p25.1 was associated with the risk of fracture (OR 32.58, 95% CI 3.95 to 1488.89; p = 8.69 × 10(-5)). We performed an in silico meta-analysis in four studies with CNV microarray data and the association with fracture risk was replicated (OR 3.11, 95% CI 1.01 to 8.22; p = 0.02). The prevalence of this deletion showed geographic diversity, being absent in additional samples from Australia, Canada, Poland, Iceland, Denmark, and Sweden, but present in the Netherlands (0.34%), Spain (0.33%), USA (0.23%), England (0.15%), Scotland (0.10%), and Ireland (0.06%), with insufficient evidence for association with fracture risk. CONCLUSIONS: These results suggest that deletions in the 6p25.1 locus may predispose to higher risk of fracture in a subset of populations of European origin; larger and geographically restricted studies will be needed to confirm this regional association. This is a first step towards the evaluation of the role of rare CNVs in osteoporosis.

Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions.

The architecture of human chromosomes in interphase nuclei is still largely unknown. Microscopy studies have indicated that specific regions of chromosomes are located in close proximity to the nuclear lamina (NL). This has led to the idea that certain genomic elements may be attached to the NL, which may contribute to the spatial organization of chromosomes inside the nucleus. However, sequences in the human genome that interact with the NL in vivo have not been identified. Here we construct a high-resolution map of the interaction sites of the entire genome with NL components in human fibroblasts. This map shows that genome-lamina interactions occur through more than 1,300 sharply defined large domains 0.1-10 megabases in size. These lamina-associated domains (LADs) are typified by low gene-expression levels, indicating that LADs represent a repressive chromatin environment. The borders of LADs are demarcated by the insulator protein CTCF, by promoters that are oriented away from LADs, or by CpG islands, suggesting possible mechanisms of LAD confinement. Taken together, these results demonstrate that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus.

Identification of Rare Variants Involved in High Myopia Unraveled by Whole Genome Sequencing.

Purpose: Myopia (nearsightedness) is a condition in which a refractive error (RE) affects vision. Although common variants explain part of the genetic predisposition (18%), most of the estimated 70% heritability is missing. Here, we investigate the contribution of rare genetic variation because this might explain more of the missing heritability in the more severe forms of myopia. In particular, high myopia can lead to blindness and has a tremendous impact on a patient and at the societal level. The exact molecular mechanisms behind this condition are not yet completely unraveled, but whole genome sequencing (WGS) studies have the potential to identify novel (rare) disease genes, explaining the high heritability. Design: Cross-sectional study performed in the Netherlands. Participants: We investigated 159 European patients with high myopia (RE > -10 diopters). Methods: We performed WGS using a stepwise filtering approach and burden analysis. The contribution of common variants was calculated as a genetic risk score (GRS). Main Outcome Measures: Rare variant burden, GRS. Results: In 25% (n = 40) of these patients, there was a high (> 75th percentile) contribution of common predisposing variants; that is, these participants had higher GRSs. In 7 of the remaining 119 patients (6%), deleterious variants in genes associated with known (ocular) disorders, such as retinal dystrophy disease (prominin 1 [PROM1]) or ocular development (ATP binding cassette subfamily B member 6 [ABCB6], TGFB induced factor homeobox 1 [TGIF1]), were identified. Furthermore, without using a gene panel, we identified a high burden of rare variants in 8 novel genes associated with myopia. The genes heparan sulfate 6-O-sulfotransferase 1 (HS6ST1) (proportion in study population vs. the Genome Aggregation Database (GnomAD) 0.14 vs. 0.03, P = 4.22E-17), RNA binding motif protein 20 (RBM20) (0.15 vs. 0.06, P = 4.98E-05), and MAP7 domain containing 1 (MAP7D1) (0.19 vs. 0.06, P = 1.16E-10) were involved in the Wnt signaling cascade, melatonin degradation, and ocular development and showed most biologically plausible associations. Conclusions: We found different contributions of common and rare variants in low and high grade myopia. Using WGS, we identified some interesting candidate genes that could explain the high myopia phenotype in some patients. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Implication of long-distance regulation of the HOXA cluster in a patient with postaxial polydactyly.

Apparently balanced chromosomal inversions may lead to disruption of developmentally important genes at the breakpoints of the inversion, causing congenital malformations. Characterization of such inversions may therefore lead to new insights in human development. Here, we report on a de novo inversion of chromosome 7 (p15.2q36.3) in a patient with postaxial polysyndactyly. The breakpoints do not disrupt likely candidate genes for the limb phenotype observed in the patient. However, on the p-arm the breakpoint separates the HOXA cluster from a gene desert containing several conserved noncoding elements, suggesting that a disruption of a cis-regulatory circuit of the HOXA cluster could be the underlying cause of the phenotype in this patient.

Infantile hypertrophic pyloric stenosis in patients with esophageal atresia.

BACKGROUND: Patients born with esophageal atresia (EA) have a higher incidence of infantile hypertrophic pyloric stenosis (IHPS), suggestive of a relationship. A shared etiology makes sense from a developmental perspective as both affected structures are foregut derived. A genetic component has been described for both conditions as single entities and EA and IHPS are variable components in several monogenetic syndromes. We hypothesized that defects disturbing foregut morphogenesis are responsible for this combination of malformations. METHODS: We investigated the genetic variation of 15 patients with both EA and IHPS with unaffected parents using exome sequencing and SNP array-based genotyping, and compared the results to mouse transcriptome data of the developing foregut. RESULTS: We did not identify putatively deleterious de novo mutations or recessive variants. However, we detected rare inherited variants in EA or IHPS disease genes or in genes important in foregut morphogenesis, expressed at the proper developmental time-points. Two pathways were significantly enriched (p < 1 × 10-5 ): proliferation and differentiation of smooth muscle cells and self-renewal of satellite cells. CONCLUSIONS: None of our findings could fully explain the combination of abnormalities on its own, which makes complex inheritance the most plausible genetic explanation, most likely in combination with mechanical and/or environmental factors. As we did not find one defining monogenetic cause for the EA/IHPS phenotype, the impact of the corrective surgery could should be further investigated.

Multiparameter Investigation of a 46,XX/46,XY Tetragametic Chimeric Phenotypical Male Patient with Bilateral Scrotal Ovotestes and Ovulatory Activity.

We report on an adult male initially presenting with gynecomastia and a painless scrotal mass without additional genital anomalies. Hyperpigmentation of the skin following the Blaschko's lines was identified. He underwent gonadectomy because of suspected cancer. Histological analyses revealed an ovotestis with ovulatory activity confirmed by immunohistochemistry with multiple markers. Karyotyping of cultured peripheral blood lymphocytes and a buccal smear revealed a 46,XX/46,XY chimeric constitution with different percentages. Multiple molecular analyses as well as blood typing implied a tetragametic origin. After the unilateral gonadectomy, the patient developed recurrent painful cystic swellings of the remaining gonad. Because of the wish to preserve hormonal activity as well as future fertility, the patient underwent surgical resection of a cystic gonadal area. The removed tissue showed ovulation-related features in addition to both testicular and ovarian tissue, diagnosed as an ovotestis. Testosterone therapy was initiated to suppress the persistently elevated gonadotropins and thereby suppress ovarian activity. During treatment, the recurrent pain complaints and cystic swellings ceased, although gonadotropin levels were not fully suppressed. Based on these observations, the importance of a detailed genetic and pathological diagnosis and the clinical dilemmas including the pros and cons of personalized treatment with gonadal preservative surgery are discussed.

A familial inverted duplication 2q33-q34 identified and delineated by multiple cytogenetic techniques.

We describe a unique family with two children having a delay in psychomotor development. In both children we identified an interstitial duplication dup(2)(q34q33) using multiple, complementary molecular cytogenetic techniques. Comparative genomic hybridisation (CGH) and array-CGH were used to determine the size and the location of the duplicated region, the orientation of the duplicated region was identified with fluorescence in situ hybridisation (FISH). Both parents demonstrated a normal karyotype and normal CGH and array-CGH-profiles. However, FISH on peripheral blood cells from the mother showed the inv dup(2) in 9% of metaphases and 19% of interphase nuclei. To our knowledge this is the first report of a mosaic carrier of duplication in the long arm of chromosome 2. The finding of chromosomal mosaicism of at least 19% in the mother increases the recurrence risk. The exact characterisation of the inv dup(2) with FISH probes enabled us to offer a reliable prenatal FISH test. Comparison of the clinical features of the two children with those of previously described cases supports the hypothesis that the characteristic facial phenotype is linked to the distal part of the 2q33-q37 region. This report illustrates that in case of two sibs with an identical structural chromosomal abnormality the possibility of parental chromosomal mosaicism must be thoroughly investigated.

Postzygotic telomere capture causes segmental UPD, duplication and deletion of chromosome 8p in a patient with intellectual disability and obesity.

Using SNP array and FISH analysis, a patient with moderate intellectual disability and obesity was found to harbour an atypical 1.6 Mb inverted duplication on 8p23.1, directly flanked by a distally located interstitial deletion of 2.3 Mb and a terminal segmental uniparental disomy. The duplicated and deleted regions lie exactly between the two segmental duplication regions. These segmental duplications on chromosome 8p23.1 are known to be involved in chromosomal rearrangements because of mutual homology and homology to other genomic regions. Genomic instability mediated by these segmental duplications is generally caused by non-allelic homologous recombination, resulting in deletions, reciprocal duplications, inversions and translocations. Additional analysis of the parental origin of the fragments of this atypical inverted duplication/interstitial deletion shows paternal contribution in the maternal derivate chromosome 8. Combined with the finding that the normal chromosome 8 carries an inversion in 8p23.1 we hypothesize that a double strand break in 8p23.1 of the maternal chromosome was postzygotically repaired with the paternal inverted copy resulting in a duplication, deletion and segmental uniparental disomy, with no particular mediation of the 8p23.1 segmental duplication regions in recombination.

Complex MAX Rearrangement in a Family With Malignant Pheochromocytoma, Renal Oncocytoma, and Erythrocytosis.

CONTEXT: Familial pheochromocytoma (PCC) has been associated with germline mutations in 16 genes. Here we investigated three siblings presenting with bilateral pheochromocytomas. In addition, the index patient also exhibited renal oncocytoma and erythrocytosis, whereas the second sibling presented with a lymph node metastasis. DESIGN: First, single-nucleotide polymorphism array and exome sequencing were performed on germline and PCC-derived DNA to identify genomic alterations in the index patient. Second, alterations were confirmed and validated by Sanger sequencing, analyzed by (multiplexed) PCR to determine the loss of the wild-type allele, and investigated by immunohistochemistry in the tumors of the three siblings. RESULTS: The index patient's germline DNA revealed a large complex genomic alteration encompassing the intragenic and promoter regions of Myc-associated factor X (MAX) and alpha-(1,6)-fucosyltransferase (FUT8). In all three siblings the MAX alteration was confirmed, and the loss of the wild-type MAX and FUT8 alleles was demonstrated in all tumors. Uniparental disomy of chromosome 14q, previously demonstrated as a hallmark for MAX-related PCC, was shown in the index patient's PCC by single-nucleotide polymorphism array. Loss of MAX and FUT8 protein expression was demonstrated by immunohistochemistry in the tumors from the three siblings. CONCLUSIONS: Our results indicate that large genomic deletions of MAX should be considered in familial and bilateral PCC with prior negative testing for gene mutations. In addition, our results confirm that MAX is a tumor suppressor gene for renal oncocytomas.

Copy number variations in 375 patients with oesophageal atresia and/or tracheoesophageal fistula.

Oesophageal atresia (OA) with or without tracheoesophageal fistula (TOF) are rare anatomical congenital malformations whose cause is unknown in over 90% of patients. A genetic background is suggested, and among the reported genetic defects are copy number variations (CNVs). We hypothesized that CNVs contribute to OA/TOF development. Quantifying their prevalence could aid in genetic diagnosis and clinical care strategies. Therefore, we profiled 375 patients in a combined Dutch, American and German cohort via genomic microarray and compared the CNV profiles with their unaffected parents and published control cohorts. We identified 167 rare CNVs containing genes (frequency<0.0005 in our in-house cohort). Eight rare CNVs - in six patients - were de novo, including one CNV previously associated with oesophageal disease. (hg19 chr7:g.(143820444_143839360)_(159119486_159138663)del) 1.55% of isolated OA/TOF patients and 1.62% of patients with additional congenital anomalies had de novo CNVs. Furthermore, three (15q13.3, 16p13.3 and 22q11.2) susceptibility loci were identified based on their overlap with known OA/TOF-associated CNV syndromes and overlap with loci in published CNV association case-control studies in developmental delay. Our study suggests that CNVs contribute to OA/TOF development. In addition to the identified likely deleterious de novo CNVs, we detected 167 rare CNVs. Although not directly disease-causing, these CNVs might be of interest, as they can act as a modifier in a multiple hit model, or as the second hit in a recessive condition.

Structural and numerical changes of chromosome X in patients with esophageal atresia.

Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) is a relatively common birth defect often associated with additional congenital anomalies such as vertebral, anal, cardiovascular, renal and limb defects, the so-called VACTERL association. Yet, little is known about the causal genetic factors. Rare case reports of gastrointestinal anomalies in children with triple X syndrome prompted us to survey the incidence of structural and numerical changes of chromosome X in patients with EA/TEF. All available (n=269) karyotypes of our large (321) EA/TEF patient cohort were evaluated for X-chromosome anomalies. If sufficient DNA material was available, we determined genome-wide copy number profiles with SNP array and identified subtelomeric aberrations on the difficult to profile PAR1 region using telomere-multiplex ligation-dependent probe amplification. In addition, we investigated X-chromosome inactivation (XCI) patterns and mode of inheritance of detected aberrations in selected patients. Three EA/TEF patients had an additional maternally inherited X chromosome. These three female patients had normal random XCI patterns. Two male EA/TEF patients had small inherited duplications of the XY-linked SHOX (Short stature HOmeoboX-containing) locus. Patients were small for gestational age at birth (

5q11.2 deletion in a patient with tracheal agenesis.

Tracheal agenesis (TA) is a rare congenital anomaly of the respiratory tract. Many patients have associated anomalies, suggesting a syndromal phenotype. In a cohort of 12 patients, we aimed to detect copy number variations. In addition to routine cytogenetic analysis, we applied oligonucleotide array comparative genomic hybridization. Our patient cohort showed various copy number variations, of which many were parentally inherited variants. One patient had, in addition to an inherited 16p12.1 deletion, a 3.6 Mb deletion on chromosomal locus 5q11.2. This patient had a syndromic phenotype, including vertebral, anal, cardiovascular and tracheo-oesophageal associated anomalies, and other foregut-related anomalies, such as cartilage rings in the oesophagus and an aberrant right bronchus. No common deletions or duplications are found in our cohort, suggesting that TA is a genetically heterogeneous disorder.

CNTNAP2 is disrupted in a family with Gilles de la Tourette syndrome and obsessive compulsive disorder.

Gilles de la Tourette syndrome (GTS) is a sporadic or inherited complex neuropsychiatric disorder characterized by involuntary motor and vocal tics. There is comorbidity with disorders like obsessive compulsive disorder and attention deficit hyperactivity disorder. Until now linkage analysis has pointed to a number of chromosomal locations, but has failed to identify a clear candidate gene(s). We have investigated a GTS family with a complex chromosomal insertion/translocation involving chromosomes 2 and 7. The affected father [46,XY,inv(2) (p23q22),ins(7;2) (q35-q36;p21p23)] and two affected children [46,XX,der(7)ins(7;2)(q35-q36;p21p23) and 46,XY,der(7)ins(7;2)(q35-q36;p213p23)] share a chromosome 2p21-p23 insertion on chromosome 7q35-q36, thereby interrupting the contactin-associated protein 2 gene (CNTNAP2). This gene encodes a membrane protein located in a specific compartment at the nodes of Ranvier of axons. We hypothesize that disruption or decreased expression of CNTNAP2 could lead to a disturbed distribution of the K(+) channels in the nervous system, thereby influencing conduction and/or repolarization of action potentials, causing unwanted actions or movements in GTS.

Isolated postaxial polydactyly type B with mosaicism of a submicroscopic unbalanced translocation leading to an extended phenotype in offspring.

Postaxial polydactyly (PAP) is characterized by the presence of one or more extra ulnar or fibular digits or parts of it. PAP type B presents frequently as a skin tag on the hand(s). It is usually an isolated malformation, but in 6.6% it is associated with other congenital abnormalities, mostly well recognizable syndromes. We present a male with PAP-B only and his daughter with an extended phenotype including mental retardation and minor dysmorphisms. Both share a cytogenetically balanced t(4;7)(p15.2;q35), present in mosaicism in the father. We found microdeletions associated with the breakpoints. The chromosomal regions described here have not been previously associated with the PAP-B phenotype. We present the first case of an individual with isolated PAP-B and a submicroscopic chromosome abnormality.

Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay.

Amplification of the proto-oncogene MYCN is a strong adverse prognostic factor in neuroblastoma patients in all tumor stages. The status of the MYCN gene has become an important factor in clinical decision making and therapy stratification. Consequently, fast and accurate assessment of MYCN gene copy number is of the utmost importance and the use of two independent methods to determine MYCN status is recommended. For these reasons we have developed and evaluated a real-time quantitative PCR (Q-PCR) assay as an alternative for time-consuming Southern blot analysis (SB), and as a second independent technique in parallel with fluorescence in situ hybridization (FISH) analysis. Advantages of Q-PCR are a large dynamic range of quantification, no requirement for post-PCR sample handling and the need for very small amounts of starting material. The accuracy of the assay was illustrated by measurement of MYCN single gene copy changes in DNA samples of two patients with 2p deletion and duplication, respectively. Two different detection chemistries i.e., a sequence specific TaqMan probe and a generic DNA binding dye SYBR Green I were evaluated and shown to yield similar results. Also, two different calculation methods for copy number determination were used i.e., the kinetic method and the comparative C(T) method, and shown to be equivalent. In total, 175 neuroblastoma samples with known MYCN status, as determined by FISH and/or SB, were examined. Q-PCR data were highly concordant with FISH and SB data. In addition to MYCN copy number evaluation, DDX1 and NAG gene copy numbers were determined using a similar Q-PCR strategy. Survival analysis pointed out that DDX1 and/or NAG amplification has no additional adverse effect on prognosis.

High-throughput analysis of subtelomeric chromosome rearrangements by use of array-based comparative genomic hybridization.

Telomeric chromosome rearrangements may cause mental retardation, congenital anomalies, and miscarriages. Automated detection of subtle deletions or duplications involving telomeres is essential for high-throughput diagnosis, but impossible when conventional cytogenetic methods are used. Array-based comparative genomic hybridization (CGH) allows high-resolution screening of copy number abnormalities by hybridizing differentially labeled test and reference genomes to arrays of robotically spotted clones. To assess the applicability of this technique in the diagnosis of (sub)telomeric imbalances, we here describe a blinded study, in which DNA from 20 patients with known cytogenetic abnormalities involving one or more telomeres was hybridized to an array containing a validated set of human-chromosome-specific (sub)telomere probes. Single-copy-number gains and losses were accurately detected on these arrays, and an excellent concordance between the original cytogenetic diagnosis and the array-based CGH diagnosis was obtained by use of a single hybridization. In addition to the previously identified cytogenetic changes, array-based CGH revealed additional telomere rearrangements in 3 of the 20 patients studied. The robustness and simplicity of this array-based telomere copy-number screening make it highly suited for introduction into the clinic as a rapid and sensitive automated diagnostic procedure.